The overall goal of this proposal is to clarify the mechanisms underlying end-stage renal disease that occurs as a result from aging and hyperhomocysteinemia (hHcys). While there are many factors contributing to elevated plasma homocysteine (Hcys) such as sex, genetics, and kidney function, age itself is a major contributor, where upwards of 30% of the elderly suffer from hHcys. We have most recently demonstrated the importance of inflammasome activation in Hcys-induced glomerular damage; however, the precise mechanisms mediating this effect remain unknown. The inflammasome, a novel multiprotein complex, has been shown to play an essential role in the early initiation of the innate immune system. Aside from immune diseases, activation of the inflammasome has recently been linked to non-immune, metabolic diseases such as diabetes, gout, silicosis, and acetaminophen-induced liver toxicity. This grant proposal hypothesizes that elevated extracellular Hcys concentrations activate NADPH oxidase (Nox) to produce reactive oxygen species (ROS) and thereby activate NALP3 inflammasomes in podocytes and increase the downstream recruitment of inflammatory cells in glomeruli, resulting in podocyte injury, local inflammation and ultimately glomerular sclerosis. To test this hypothesis, three specific aims are proposed. Specific Aim 1 will determine whether the in vitro formation and activation of NALP3 inflammasomes by Hcys in mouse podocytes are associated with Nox-mediated redox signaling and will seek to explore the mechanisms mediating the precise action of Nox-derived ROS on inflammasome activation. Specific Aim 2 will attempt to test whether in vivo hHcys-induced activation of NALP3 inflammasomes and associated glomerular injury are blocked by selective inhibition of local Nox activity and expression or by knocking out the gp91phox gene in mice. Specific Aim 3 will determine whether in vivo pharmacological activation of Nox and overexpression of gp91phox gene enhance inflammasome formation in glomeruli of hyperhomocysteinemic mice and to observe whether gene rescuing in gp91phox knockout mice restores hHcys-induced activation of podocyte inflammasomes. This proposal will be the first to examine the triggering role of Nox-mediated redox signaling in inflammasome activation and the first to link both Nox and the inflammasome to the early initiating mechanisms leading to glomerular damage induced by Hcys. Results from these proposed studies may lead to the discovery of new therapeutic targets for the prevention and treatment of glomerular damage and the eventual end-stage renal disease that occurs in patients with hHcys, especially in the elderly.